Non-invasive enzyme screen for tissue remodelling-associated conditions

ABSTRACT

Methods and kits for diagnosing the presence of and prognosing the appearance of tissue remodelling-associated conditions, involving the presence of enzyme complexes in a biological sample, are disclosed. In particular, the method pertains to diagnosing the presence of or prognosing appearance of metastatic cancer by the identification of high molecular weight enzyme complexes comprising MMPs.

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisionalapplication Ser. No. 60/240,489 filed on Oct. 13, 2000, the contents ofwhich are expressly incorporated herein by reference. This applicationis also related to Serial No. 08/639,373 filed on Apr. 26, 1996,(abandoned), U.S. Pat. No. 6,037,138, and Serial No. 09/469,637(pending), the entire contents of each are expressly incorporated hereinby reference. The contents of Yan, L. et al. (2001) J. Biol.Chem. 276:37258-37265 are expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Matrix metalloproteinases (MMP) are a family of endopeptidaseswhose activities depend on metal ions, such as Zn⁺⁺ and Ca⁺⁺.Collectively, MMPs are capable of degrading all the molecular componentsof extracellular matrix (ECM), the barrier separating the tumor cellsfrom the normal surrounding tissues, which is disassembled as part ofthe metastatic process (Lochter, A., et al. (1998) Ann N Y Acad Sci.857: 180-93). MMPs have been shown to play important roles in a varietyof biological as well as pathological processes, especially in tumorcell invasion and metastasis (Kleiner, D. E. and Stetler-Stevenson, W.G. (1999) Cancer Chemother Pharmacol. 43: S42-51). Overproduction ofMMPs by tumor cells or surrounding stromal cells has been correlatedwith the metastatic phenotype. In particular, U.S. Ser. No. 09/469,637,the contents of which are herein incorporated by reference in theirentirety, teaches that intact and biologically active MMPs can bedetected in biological samples of cancer patients and are independentpredictors of disease status. The MMP activities detected in U.S. Ser.No. 09/469,637 include, for example, MMP-9 (92 kDa, gelatinase B, typeIV collagenase, EC3.4.24.35) and MMP-2 (72 kDa, gelatinase A, type IVcollagenase, EC3.4.24.24). Both of these MMPs have been shown to beindependent predictors of tissue remodeling-associated conditions, e.g.,cancer. In addition to these two major gelatinase species, several MMPactivities with molecular sizes of equal to, or greater than, 150 kDawere observed and were referred to as high molecular weight (hMW) MMPs.Elevated MMP levels in biological fluids, including serum, plasma, andurine from animals bearing experimental tumors or from cancer patientshave also been reported in several other studies (Nakajima, M., et al.,(1993) Cancer Res. 53: 5802-7; Zucker, S., et al. (1994) Ann N Y AcadSci. 732: 248-62; Baker, T., et al. (1994) Br J Cancer. 70: 506-12;Garbisa, S., et al. (1992) Cancer Res. 52: 4548-9, 1992).

SUMMARY OF THE INVENTION

[0003] With the advances in cancer therapies, early diagnosis and/orprognosis are becoming increasingly important for the disease outcome.Accordingly, the present invention characterizes the molecular identityof hMW MMPs found in biological samples of subjects diagnosed withtissue remodelling-associated diseases, e.g., cancer, and provides earlydiagnosis/prognosis of such diseases. With the identification of thesehMW MMPS, e.g., high molecular weight enzyme complexes, the presentinvention facilitates the development of non-invasive diagnostic and/orprognostic methods to predict tissue remodelling-associated diseases,such as cancer.

[0004] The present invention provides methods and kits for detectingbiological markers, e.g., high molecular weight enzyme complexes, tonon-invasively monitor the diagnosis and prognosis of tissueremodelling-associated conditions, e.g., cancers. Tissueremodelling-associated conditions encompassed by such methods includediseases such as prostate cancer, breast cancer, ovarian cancer, braintumors, arthritic conditions, obstructive conditions, and ulcerativeconditions. The methods of the instant invention use biological fluidsamples, e.g., urine samples, that may be obtained by personnel withoutmedical training, and do not require visiting a clinic or hospital. Thestatistical association between positive results and occurrence oftissue remodelling-associated conditions are applied to early diagnosesof the appearance of these conditions, and to prognoses of changes inthese conditions.

[0005] In one embodiment, the present invention provides non-invasivemethods for facilitating the diagnosis of a subject for a tissueremodelling-associated condition. Such methods include obtaining abiological sample from a subject, and detecting a high molecular weightenzyme complex in the biological sample. The methods further includecorrelating the presence or absence of the high molecular weight enzymecomplex with the presence or absence of a tissue remodelling-associatedcondition, thereby facilitating the diagnosis of the subject for atissue remodelling-associated condition.

[0006] In another embodiment, the tissue remodelling-associatedcondition is cancer, e.g., organ-confined prostate cancer, metastaticprostate cancer, cancer found in cells of epithelial origin, mesodermalorigin, endodermal origin or hematopoietic origin, and cancer selectedfrom the group consisting of cancers of the nervous system, breast,retina, lung, skin, kidney, liver, pancreas, genito-urinary tract, andgastrointestinal tract. In another embodiment, the tissueremodelling-associated condition is an arthritic condition, anobstructive condition, or a degenerative condition.

[0007] In still another embodiment, the high molecular weight enzymecomplex comprises a protease, e.g., a serine protease, e.g., a matrixmetalloproteinase, e.g., an MMP-9.

[0008] In yet another embodiment, the high molecular weight enzymecomplex further comprises a lipocalin, e.g., NGAL, and/or a TIMP, e.g.,TIMP-1.

[0009] In still another embodiment, the high molecular weight enzymecomplex comprises an enzyme complexed with itself to form a multimer,e.g., a dimer or a trimer. Such a multimer can further be complexed witha lipocalin, e.g., NGAL, and/or a TIMP, e.g., TIMP-1.

[0010] In still yet another embodiment, the molecular weight of the highmolecular weight enzyme complex is at least about 115 kDa to at leastabout 125 kDa. In another embodiment, the molecular weight of the highmolecular weight enzyme complex is at least about 150 kDa.

[0011] In another embodiment, the methods of the present inventioninclude obtaining a biological sample from a subject and detectinglipocalin in the biological sample. Such methods further includecorrelating the presence or absence of the lipocalin with the presenceor absence of a tissue remodelling-associated condition, therebyfacilitating the diagnosis of the subject for a tissueremodelling-associated condition.

[0012] In still another embodiment, the present invention provides kitsfor facilitating the diagnosis and prognosis of a tissueremodelling-associated condition. Such kits include a container having areagent for detecting a high molecular weight enzyme complex in abiological sample and instructions for using the reagent for detectingthe high molecular weight enzyme complex which facilitates the diagnosisand prognosis of a tissue remodelling-associated condition.

DESCRIPTION OF THE FIGURES

[0013]FIG. 1 shows a substrate gel electrophoresis and NGAL Western blotanalysis of urine samples. A. Substrate gel electrophoresis of MMPs inurine samples: 50 μl of untreated urine samples were analyzed for MMPactivities. Four major gelatinase activities were detected with apparentmolecular masses of approximately 200,000, 125,000, 92,000, and 72,000.Their identities are marked with arrows on right. The molecular sizemarkers are Perfect Protein Markers (Novagen, Madison, Wis.) with sizesof 150 kDa, 100 kDa, 75 kDa, and 50 kDa (arrows on left). B. 20 μg ofconcentrated urine samples were separated on a 4-15% SDS-PAGE gel undernon-reducing conditions. Western blot analysis was carried out using apolyclonal antibody against human NGAL. The molecular size markers areKaleidoscope Prestained Standards (Bio-Rad, Hercules, Calif.) with sizesof 126 kDa, 90 kDa, 44 kDa, 34 kDa, and 17 kDa (arrows on left).

[0014]FIG. 2 shows a Western blot and substrate gel electrophoresis ofurine samples and purified human neutrophil MMP-9/NGAL complex. A. NGALWestern blot analysis: A concentrated urine sample containing the 125kDa MMP activity, together with purified human neutrophil MMP-9/NGAL,were separated by 4-15% SDS-gel electrophoresis under non-reducingconditions, and subsequently subjected to Western blot analysis using apolyclonal antibody against human NGAL. The 125 kDa MMP-9/NGAL complexis marked (arrow on right). B. Substrate gel electrophoresis: The sameurine sample and purified human neutrophil MMP-9/NGAL complex wereanalyzed with substrate gel electrophoresis. The positions of MMP-9dimer (200 kDa), MMP-9/NGAL (125 kDa), MMP-9 (92 kDa), and MMP-2 (72kDa) are denoted with arrows on right. The molecular size markers arePerfect Protein Markers (Novagen, Madison, Wis.) with sizes of 150 kDa,100 kDa, 75 kDa, and 50 kDa (arrows on left).

[0015]FIG. 3 shows an immunoprecipitation of the 125 kDa MMP activityusing anti-NGAL antibody. 50 μl of urine samples (1:1 v/v diluted withRIPA) containing the 125 kDa MMP activity were mixed with 1.0, 0.1 or0.01 μl of anti-NGAL antibody or a control antibody. After incubating onice for thirty minutes, the antibody-antigen complexes were removedusing Zysorbin. The supernatants were subjected to substrate gelelectrophoresis to detect the remaining MMP activities. The increasedMMP-2 activity observed in the sample treated with 1.0 μl of the controlantibody was the endogenous MMP-2 activity from the serum.

[0016]FIG. 4 shows reconstitution of MMP-9/NGAL complexes in vitro. A.Recombinant human MMP-9 and NGAL were diluted in gelatinase buffers withdifferent pH values and were subsequently mixed in a molar ratio of 1:10(proMMP-9 to NGAL). In vitro reconstitution was carried out at 37° C.for one hour. 10 μM proMMP-9 was loaded in each lane. Purified humanneutrophil MMP-9/NGAL was included as a control. B. Recombinant humanMMP-9 and NGAL were diluted in normal urine containing no MMP activitiesand were subsequently mixed in different molar ratios (proMMP-9 toNGAL=2:1, 1:5, 1:10, 1:20). After one hour incubation at 37° C.,MMP-9/NGAL complex formation was analyzed using substrate gelelectrophoresis. The positions of the 125 kDa and 115 kDa MMP-9/NGALactivity are respectively denoted with the arrow and the arrowhead onright. The molecular size markers are Perfect Protein Markers (Novagen,Madison, Wis.) with sizes of 150 kDa, 100 kDa, 75 kDa, and 50 kDa(arrows on left).

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention features non-invasive methods forfacilitating the diagnosis of a subject for a tissueremodelling-associated condition (TRAC), especially cancers, obstructiveand degenerative conditions, and arthritic conditions. Detection of apattern of enzyme complexes, e.g., high molecular weight (hMW) enzymecomplexes, in a biological sample from a subject is used to facilitatediagnosis and prognosis of a TRAC.

[0018] The language “high molecular weight enzyme complex” includes anenzyme associated with or bound to another molecule wherein the complexhas a high molecular weight allowing it to be used for its intendedfunction of the present invention. Examples of enzyme complexes include,among others, an enzyme bound to another enzyme, an enzyme bound to anenzyme inhibitor, and an enzyme bound to a protein binding molecule,e.g., a lipocalin. Enzyme complexes which comprise enzymes bound tothemselves, e.g., multimers, e.g., dimers and trimers, are alsoencompassed by the present invention.

[0019] High molecular weight enzyme complexes include enzyme complexeswhich have a molecular weight of at least about 115 kDa, e.g., at leastabout 120 kDa, e.g., at least about 125 kDa, e.g., at least about 130kDa, e.g., at least about 135 kDa, and, e.g., at least about 140 kDa.High molecular weight enzyme complexes which have a molecular weight ofat least about 145 kDa, e.g., at least about 150 kDa, and greater than150 kDa are also included.

[0020] The ranges of high molecular weight values intermediate to thoselisted also are intended to be part of this invention, e.g. at leastabout 115 kDa to at least about 120 kDa, at least about 120 kDa to atleast about 125 kDa, at least about 125 kDa to at least about 130 kDa,at least about 130 kDa to at least about 135 kDa, at least about 135 kDato at least about 140 kDa, at least about 140 kDa to at least about 145kDa, and at least about 145 kDa to at least about 150 kDa. For example,ranges of high molecular weight values using a combination of any of theabove values recited as upper and/or lower limits are intended to beincluded.

[0021] In one embodiment of the invention, the high molecular weightenzyme complex does not have a molecular weight of 115 kDa. In anotherembodiment, the high molecular weight enzyme complex does not includeNGAL. In another embodiment, the high molecular weight enzyme complexdoes not include a progelatinase B enzyme. In yet another embodiment ofthe invention, the high molecular weight enzyme complex does not includea progelatinase B enzyme associated with NGAL.

[0022] The term “enzyme” is art recognized and includes proteincatalysts of chemical reactions. Enzymes can be a whole intact enzyme orportions or fragments thereof. The enzymes encompassed by the enzymecomplexes of the current invention include naturally occurring enzymesthat catalytically degrade proteins, i.e. the enzymes known as proteasesor proteinases. By proteinase is meant a progressive exopeptidase thatdigest proteins by removing amino acid residues from either the Nterminal or C terminal which reaction proceeds to achieve significantdegradation, or an endopeptidase which destroys the amide bond betweenamino acid residues with varying degrees of residue specificity. Theterm “protease” may also include the highly specific amino acidpeptidases that remove a single amino acid from an N terminus or Cterminus of a protein. Examples are alanine aminopeptidase (EC 3.4.11.2)and leucine aminopeptidase (EC 3.4.11.1), which remove alanine orleucine, respectively, from the amino terminus of a protein that mayhave alanine and leucine, respectfully, at the amino terminus. Themolecular weights of the enzymes comprising the enzyme complexes of theinvention include, but not limited to, molecular weights in the range ofapproximately 72 kDa, approximately 92 kDa, approximately 115 kDa toapproximately 125 kDa and approximately 150 kDa or greater. The term“enzyme” includes polymorphic variants that are silent mutationsnaturally found within the human population.

[0023] In one embodiment, the enzyme complexes of the present inventioncomprise proteases or proteinases. The term proteases (and itsequivalent term proteinases) is intended to include those endopeptidasesand progressive exopeptidases that are capable of substantially reducingthe molecular weight of the substrate and destroying its biologicalfunction, especially if that biological function of the substrate is tobe a structural component of a matrix barrier. Amino acid peptidasessuch as alanine aminopeptidase and leucine aminopeptidase are alsobroadly included among proteases, however do not share the property ofsignificantly reducing the molecular weight of the substrate protein.

[0024] Many thousands of proteases occur naturally, and each may appearat different times of development and in different locations in anorganism. The invention herein features enzymes of the class of thematrix metalloproteinases (MMPs, class EC 3.4.24). These enzymes, whichrequire a divalent cation for activity, are normally expressed early inthe development of the embryo, for example, during hatching of an zygotefrom the zona pellucida, and again during the process of attachment ofthe developing embryo to the inside of the uterine wall. Enzymeactivities such as N-acetylglucosaminidase (EC 3.2.1.50) appear in urinein the case of renal tubular damage, for example, due to diabetes (Carr,M. (1994) J. Urol. 151(2):442-445; Jones, A., et. al. (1995) Annals.Clin. Biochem., 32:68-62). That these activities appear in urine as aresult of renal tubular damage is irrelevant to the present invention asdescribed herein.

[0025] The term “matrix-digesting enzyme” includes an enzyme capable ofdigesting or degrading a matrix, e.g., a mixture of proteins andproteoglycans that comprise a layer in a tissue on which certain typesof cells are found. Matrix-digesting enzymes are expressed during stagesof normal embryogenesis, pregnancy and other processes involving tissueremodelling. In addition, some of these enzymes, for example some matrixmetalloproteinases (MMPs), degrade the large extracellular matrixproteins of the parenchymal and vascular basement membranes that serveas mechanical barriers to tumor cell migration. These MMPs are producedin certain cancers and are associated with metastasis (Liotta, L. A., etal. (1991) Cell 64:327-336). Examples of MMPs are the type IVcollagenases, e.g., MMP-2 (gelatinase A. EC 3.4.24.24) and MMP-9(gelatinase B, 3.4.24.35), and stromelysins (EC 3.4.24.17 and3.4.24.22). Some MMPs are specifically inhibited by molecules calledtissue inhibitors of metalloproteinases (TIMPs, Woessner, J. F., Jr.(1995) Ann. New York Acad. Sci., 732:11-21),which also may be overproduced by tumor cells, however under certain conditions enzymeactivity is in molar excess over the TIMPs (Freeman, M. R. et al. (1993)J. Urol. 149:659; Lu, X. et al. (1991) Cancer Res.51:6231-6235;Kossakowska, A. E. et al. (1991) Blood 77:2475-2481).Accordingly, in one embodiment of this invention, the enzyme complexesof the present methods comprise an inhibitor of the enzyme (TIMPS, e.g.,TIMP-1 or TIMP-2). The detection of an inhibitor can be accomplishedusing art-recognized techniques. Many of MMPs are translated asproenzymes, and may be found in a variety of structures, with ranges ofmolecular weights including smaller forms (45 kDa, 55 kDa, 62 kDa), andlarger forms (72 kDa, 82 kDa, 92 kDa, and higher polymers such as 150kDa and greater).

[0026] In another embodiment of the invention, the high molecular weightenzyme complex comprises a protein binding molecule, e.g., a lipocalin.Lipocalins are small secreted proteins that bind small, hydrophobicmolecules to form molecular complexes. Lipocalins are implicated in avariety of functions including, among others, regulation of the immuneresponse, e.g., lipocalins can exert certain immunomodulatory effects invitro. It has been shown that neutrophil lipocalin covalently attachesto human neutrophil gelatinase (type IV collagenase) thus formingNeutrophil Gelatinase-Associated Lipocalin (NGAL) (Treibel et al. (1992)and Kjelsen et al. (1993)) although most of the protein is secreted inuncomplexed form. These authors prose a regulatory role for NGAL on theaction of the gelatinase.

[0027] In another embodiment, the present invention includes methods ofdetecting a lipocalin, e.g., NGAL, as an indicator of a TRAC. Suchlipocalins can be detected in a biological sample as an isolatedlipocalin or as multimers of lipocalins, e.g., dimers and trimers.

[0028] The tissue remodelling conditions that can be monitored by themethods of this invention include a variety of types of cancer;moreover, the enzymes are suitable for diagnosis of other tissueremodelling conditions, such as arthritis, degenerative conditions, andobstructive conditions. The invention provides non-invasive methods fordiagnosing these conditions by assay for enzyme complexes, e.g., hMWenzyme complexes, in biological fluids.

[0029] The methods of this invention embody detection of enzymes inurine, for diagnosis and prognosis of cancer. The invention also relatesto diagnosis and prognosis of metastatic prostate cancer. The varietiesof cancer suitable for diagnosis by the methods of this inventioninclude, among others, cancers of epithelial origin, for example,cancers of the nervous system, breast, retina, lung, skin, kidney,liver, pancreas, genito-urinary tract, ovarian, uterine and vaginalcancers, and gastrointestinal tract cancers, which form in cells ofepithelial origin. Using the methods described here, cancers ofmesodermal and endodermal origin, for example, cancers arising in boneor in hematopoietic cells, are also diagnosed.

[0030] The term “subject,” as used herein, includes a living animal orhuman in need of diagnosis or prognosis for, or susceptible to, acondition, in particular an “tissue remodelling-associated condition” asdefined below. The subject is an organism capable of responding totissue remodelling signals such as growth factors, under somecircumstances, the subject is susceptible to cancer and to arthritis. Inone embodiment, the subject is a mammal, including humans and non-humanmammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, andmice. In one embodiment, the subject is a human. The term “subject” doesnot preclude individuals that are entirely normal with respect to tissueremodelling-associated conditions or normal in all respects. The subjectmay formerly have been treated surgically or by chemotherapy, and may beunder treatment by hormone therapy or have been treated by hormonetherapy in the past.

[0031] The term “patient,” as used herein, includes a human subject whohas presented at a clinical setting with a particular symptom orsymptoms suggesting one or more diagnoses. A patient may be in need offurther categorization by clinical procedures well-known to medicalpractitioners of the art (or may have no further disease indications andappear to be in any or all respects normal). A patient's diagnosis mayalter during the course of disease progression, such as development offurther disease symptoms, or remission of the disease, eitherspontaneously or during the course of a therapeutic regimen ortreatment. Thus, the term “diagnosis” does not preclude differentearlier or later diagnoses for any particular patient or subject. Theterm “prognosis” includes an assessment for a subject or patient of aprobability of developing a condition associated with or otherwiseindicated by presence of one or more enzymes in a biological sample,e.g., in urine.

[0032] The term “biological sample” includes biological samples obtainedfrom a subject. Examples of such samples include urine, blood taken froma prick of the finger or other source such as intravenous, bloodfractions such as serum and plasma, feces and fecal material andextracts, saliva, cerebrospinal fluid, amniotic fluid, mucus, and celland tissue material such as cheek smear, Pap smear, fine needleaspiration, sternum puncture, and any other biopsied material takenduring standard medical and open surgical procedures.

[0033] The term “invasiveness” as used here with respect to metastaticcancer (Darnell, J. (1990) Molecular Cell Biology, Third Ed., W. H.Freeman, NY) is distinct from the use of the term “invasive” to describea medical procedure, and the distinction is made in context. “Invasive”for a medical procedure pertains to the extent to which a particularprocedure interrupts the integrity of the body. “Invasiveness” rangesfrom fully non-invasive, such as collection of urine or saliva; tomildly invasive, for example a Pap smear, a cheek scrape or blood test,which requires trained personnel in a clinical setting; to moreinvasive, such as a sternum marrow collection or spinal tap; toextensively invasive, such as open surgery to detect the size and natureof tumors by biopsy of material, taken for example during brain surgery,lung surgery, or transurethral resection in the case of prostate cancer.

[0034] Cancer or neoplasia is characterized by deregulated cell growthand division. A tumor arising in a tissue originating from endoderm orexoderm is called a carcinoma, and one arising in tissue originatingfrom mesoderm is known as a sarcoma (Darnell, J. (1990) Molecular CellBiology, Third Ed., W. H. Freeman, NY). A current model of the mechanismfor the origin of a tumor is by mutation in a gene known as an oncogene,or by inactivation of a second tumor-suppressing genes (Weinberg, R. A.(September 1988) Scientific Amer. 44-51). The oncogenes identified thusfar have arisen only in somatic cells, and thus have been incapable oftransmitting their effects to the germ line of the host animal. Incontrast, mutations in tumor-suppressing genes can be identified in germline cells, and are thus transmissible to an animal's progeny. Examplesof cancers include cancers of the nervous system, breast, retina, lung,skin, kidney, liver, pancreas, genito-urinary tract, gastrointestinaltract, cancers of bone, and cancers of hematopoietic origin such asleukemias and lymphomas. In one embodiment of the present invention, thecancer is not a cancer of the bladder.

[0035] An arthritic condition such as rheumatoid arthritis is an exampleof a TRAC since the disease when chronic is characterized by disruptionof collagenous structures (J. Orten et al. (1982) Human Biochemistry,Tenth Ed., C. V. Mosby, St. Louis, Mo.). Excess collagenase is producedby cells of the proliferating synovium. Other TRAC conditions such asulcerative, obstructive and degenerative diseases are similarlycharacterized by alterations in the enzymes of metabolism of structuralproteins.

[0036] The term “electrophoresis” is used to indicate any separationsystem of molecules in an electric field, generally using an inertsupport system such as paper, starch gel, or polyacrylamide. Theelectrophoresis methods with polyacrylamide gels and the sodium dodecylsulfate denaturing detergent are described in the Examples below. Theprotocols are not intended to exclude equivalent procedures known to theskilled artisan. Other SDS polyacrylamide procedures, known to theskilled artisan, may be used, e.g., a single polyacrylamideconcentration such as 10%, may be substituted for the gradient in theseparation gel. The physical support for the electrophoretic matrix maybe capillary tubes rather than glass plates. Details of severalSDS-polyacrylamide gel electrophoresis systems are described in manyreview articles and biotechnology manuals (e.g., Maniatis, T., MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.). The method is not limited to use of SDS andother detergents. Further, electrophoresis in the absence of detergentsmay be employed. Proteins may be separated under non-denaturingconditions, for example in the presence of urea on a polyacrylamidematrix (Maniatis, supra), or by charge, for example by the procedure ofisoelectric focussing.

[0037] In using an electrophoretic technique for separation of enzymes,the electrophoretogram may be developed as a zymogram. The term“zymography” is meant here to include any separations system utilizing achemically inert separating or support matrix, that allows detection ofan enzyme following electrophoresis, by exposing the matrix of theseparations system to conditions that allow enzyme activity andsubsequent detection. More narrowly, the term zymography designatesincorporation of an appropriate substrate for the enzyme of interestinto the inert matrix, such that exposing the matrix to the conditionsof activity after the electrophoresis stop yields a system to visualizethe precise location, and hence the mobility, of the active enzyme. Bytechniques well-known to the skilled artisan, the molecular weights ofproteins are calculated based on mobilities derived from positions on azymogram. Such techniques include comparison with molecular weightstandards, the mobilities of which are determined from general proteinstains or from pre-stains specific to those standards, and comparisonwith positive controls of purified isolated enzymes of interest, whichare visualized by the technique of the zymogram, i.e., enzyme activity.

[0038] In particular, substrates for detection of proteases byzymography are included in the electrophoresis matrix. For type IVcollagenases, the natural substrate is a type IV collagen and gelatin, atype I collagen derivative, is used for the zymography substrate in theExamples presented herein. However other proteins that are suitable fordetection of further proteases of interest in TRAC diagnosis, forexample, include fibronectin; vitronectin; collagens of types I throughIII and V through XII; procollagens; elastin; laminin; plasmin;plasminogen; entactin; nidogen; syndecan; tenascin; and sulfatedproteoglycans substituted with such saccharides as hyaluronic acid,chondroitin-6-sulfate, condroitin-4-sulfate, heparan sulfate, keratansulfate, and dermatan sulfate and heparin. Further, convenientinexpensive substrate proteins such as casein, which may not be thenatural target of a protease of interest, but are technicallyappropriate, are included as suitable substrate components of thezymography techniques of the present invention. Chemically synthesizedmimetics of naturally occurring protein substrates are also potentialzymography substrates, and may even be designed to have favorableproperties, such chromogenic or fluorogenic ability to produce a coloror fluorescent change upon enzymatic cleavage.

[0039] Zymography may be adapted to detection of a protease inhibitor inthe biological sample. Since a variety of natural MMP inhibitors areelaborated, such as TIMP-1 and TIMP-2, and are found to be deregulatedduring TRAC situations, the present invention includes detection ofenzyme complexes which comprise enzyme inhibitors, e.g., TIMPs. Thus forexample, a “reporter enzyme” for which an enzyme inhibitory activity isbeing measured, may be incubated with each biological sample obtained bysubjects and patients, in one or more quantities corresponding to one ormore aliquots of sample, prior to electrophoresis. This enzyme isomitted from one aliquot of the biological sample. The inhibitorypresence in the sample is detected as disappearance or decrease of thereporter enzyme band from the developed zymogram. Alternatively,functional enzyme activity assays which include in the reaction mix aknown level of active enzyme, to which is added aliquots of experimentalsamples with putative inhibitory activity, can detect the presence ofinhibitors.

[0040] Further, the enzymes of tissue remodelling extend to enzymeactivities beyond those of proteolytic activity. For example, enzymesthat are substituted with residues such as glycosyl, phosphate, sulfate,lipids and nucleotide residues (e.g. adenyl) are well-known to thoseskilled in the art. These residues are in turn added or removed by otherenzymes, e.g., glycosidases, kinases, phosphatases, adenyl transferases,etc. Convenient detection methods for the presence of such activitiesfor TRAC diagnosis and prognosis are readily developed by those withskill in the art, and are intended to comprise part of the inventionhere.

[0041] The zymogram as described in the Examples herein is developed byuse of a general stain for protein, in this case, Coomassie Blue dye.The development is possible with general protein stains, e.g., AmidoBlack dye, and SYPRO Orange stain (Biorad Laboratories, Hercules, Calif.94537). Further, enzyme activity may be detected by additionaltechniques beyond that of a clear zone of digestion in a stained matrix,for example, by absence of areas of radioactivity with a radio-labelledsubstrate, by change in mobility of a radio-labelled substrate, or byabsence of or change in mobility of bands of fluorescence or colordevelopment with use of fluorogenic or chromogenic substrates,respectfully.

[0042] Quantitative densitometry can be performed with zymograms byplacing the gel directly on an activated plate of a Molecular Dynamicsphosphorimager (Molecular Dynamics, 928 East Arques Ave., Sunnyvale,Calif. 94086), or with a Datacopy G8 plate scanner attached to aMacIntosh computer equipped with an 8-bit videocard and McImage (XeroxImaging Systems). Background measurements, areas of the gel separatefrom sample lanes, can similarly be scanned, and values subtracted fromthe readings for enzyme activities.

[0043] Another electrophoretically-based technique for analysis of abiological sample for presence of specific proteins is an affinity-basedmobility alteration system (Lander, A. (1991) Proc. Natl. Acad. Sci.U.S., 88(7):2768-2772). An MMP or other type of enzyme of interest mightbe detected, for example, by inclusion of a substrate analog that bindsessentially irreversibly to the enzyme, hence decreasing the mobility.The affinity material is present during electrophoresis, and isincorporated into the matrix, so that detection of the enzyme ofinterest occurs as a result of alteration of mobility in contrast tomobility in the absence of the material. Yet another technique ofelectrophoretic protein separation is based on the innate charge of aprotein as a function of the pH of the buffer, so that for any proteinspecies, there exists a pH at which that protein will not migrate in anelectric field, or the isoelectric point, designated pI. Proteins of abiological sample, such as a urine sample, may be separated byisoelectric focussing, then developed by assaying for enzymatic activityfor example by transfer to material with substrate, i.e., zymography.Electrophoresis is often used as the basis of immunological detections,in which the separation step is followed by physical or electrophoretictransfer of proteins to an inert support such as paper or nylon (knownas a “blot”), and the blotted pattern of proteins may be detected by useof a specific primary binding (Western blot) by an antibody followed bydevelopment of bound antibodies by secondary antibodies bound to adetecting enzyme such as horse radish peroxidase. Additionalimmunological detection systems for TRAC enzyme complexes are nowdescribed in detail below.

[0044] The term “antibody” as used herein is intended to includefragments thereof which are also specifically reactive with one of thecomponents in the methods and kits of the invention. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described above for whole antibodies. Forexample, F(ab)₂ fragments can be generated by treating an antibody withpepsin. The resulting F(ab)₂ fragment can be treated to reduce disulfidebridges to produce Fab fragments. The term “antibody” is furtherintended to include single chain, bispecific and chimeric molecules. Theterm “antibody” includes possible use both of monoclonal and polyclonalantibodies (Ab) directed against a target, according to the requirementsof the application.

[0045] Polyclonal antibodies can be obtained by immunizing animals, forexample rabbits or goats, with a purified form of the antigen ofinterest, or a fragment of the antigen containing at least one antigenicsite. Conditions for obtaining optimal immunization of the animal, suchas use of a particular immunization schedule, and using adjuvants e.g.Freund's adjuvant, or immunogenic substituents covalently attached tothe antigen, e.g. keyhole limpet hemocyanin, to enhance the yield ofantibody titers in serum, are well-known to those in the art. Monoclonalantibodies are prepared by procedures well-known to the skilled artisan,involving obtaining clones of antibody-producing lymphocyte, i.e. celllines derived from single cell line isolates, from an animal, e.g. amouse, immunized with an antigen or antigen fragment containing aminimal number of antigenic determinants, and fusing said clone with amyeloma cell line to produce an immortalized high-yielding cell line.Many monoclonal and polyclonal antibody preparations are commerciallyavailable, and commercial service companies that offer expertise inpurifying antigens, immunizing animals, maintaining and bleeding theanimals, purifying sera and IgG fractions, or for selecting and fusingmonoclonal antibody producing cell lines, are available.

[0046] Specific high affinity binding proteins, that can be used inplace of antibodies, can be made according to methods known to those inthe art. For example, proteins that bind specific DNA sequences may beengineered (Ladner, R. C., et. al., U.S. Pat. No. 5,096,815), andproteins that bind a variety of other targets, especially proteintargets (Ladner, R. C., et. al., U.S. Pat. No. 5,233,409; Ladner, R. C.,et al., U.S. Pat. No. 5,403,484) may be engineered and used in thepresent invention for covalent linkage to a chelator molecule, so that acomplex with a radionuclide may be formed under mild conditions.Antibodies and binding proteins can be incorporated into large scalediagnostic or assay protocols that require immobilizing the compositionsof the present invention onto surfaces, for example in multi-well plateassays, or on beads for column purifications.

[0047] General techniques to be used in performing various immunoassaysare known to those of ordinary skill in the art. Moreover, a generaldescription of these procedures is provided in U.S. Pat. No. 5,051,361which is incorporated herein by reference, and by procedures known tothe skilled artisan, and described in manuals of the art (Ishikawa, E.,et. al. (1988) Enzyme Immunoassay Igaku-shoin, Tokyo, NY; Hallow, E. andD. Lane, Antibodies: A Laboratory Manual, CSH Press, NY). Examples ifseveral immunoassays are given discussed here.

[0048] Radioimmunoassays (RIA) utilizing radioactively labeled ligands,for example, antigen directly labeled with ³H, or ¹⁴C, or ¹²⁵I, measurepresence of MMP's as antigenic material. A fixed quantity of labeled MMPantigen competes with unlabeled antigen from the sample for a limitednumber of antibody binding sites. After the bound complex of labeledantigen-antibody is separated from the unbound (free) antigen, theradioactivity in the bound fraction, or free fraction, or both, isdetermined in an appropriate radiation counter. The concentration ofbound labeled antigen is inversely proportional to the concentration ofunlabeled antigen present in the sample. The antibody to MMP can be insolution, and separation of free and bound antigen MMP can beaccomplished using agents such as charcoal, or a second antibodyspecific for the animal species whose immunoglobulin contains theantibody to MMP. Alternatively, antibody to MMP can be attached to thesurface of an insoluble material, which in this case, separation ofbound and free MMP is performed by appropriate washing.

[0049] Immunoradiometric assays (IRMA) are immunoassays in which theantibody reagent is radioactively labeled. An IRMA requires theproduction of a multivalent MMP conjugate, by techniques such asconjugation to a protein e.g., rabbit serum albumin (RSA). Themultivalent MMP conjugate must have at least 2 MMP residues per moleculeand the MMP residues must be of sufficient distance apart to allowbinding by at least two antibodies to the MMP. For example, in an IRMAthe multivalent MMP conjugate can be attached to a solid surface such asa plastic sphere. Unlabeled “sample” MMP and antibody to MMP which isradioactively labeled are added to a test tube containing themultivalent MMP conjugate coated sphere. The MMP in the sample competeswith the multivalent MMP conjugate for MMP antibody binding sites. Afteran appropriate incubation period, the unbound reactants are removed bywashing and the amount of radioactivity on the solid phase isdetermined. The amount of bound radioactive antibody is inverselyproportional to the concentration of MMP in the sample.

[0050] Other immunoassay techniques use enzyme labels such ashorseradish peroxidase, alkaline phosphatase, luciferase, urease, andβ-galactosidase. For example, MMP's conjugated to horseradish peroxidasecompete with free sample MMP's for a limited number of antibodycombining sites present on antibodies to MMP attached to a solid surfacesuch as a microtiter plate. The MMP antibodies may be attached to themicrotiter plate directly, or indirectly, by first coating themicrotiter plate with multivalent MMP conjugates (coating antigens)prepared for example by conjugating MMP with serum proteins such asrabbit serum albumin (RSA). After separation of the bound labeled MMPfrom the unbound labeled MMP, the enzyme activity in the bound fractionis determined colorimetrically, for example by a multi-well microtiterplate reader, at a fixed period of time after the addition ofhorseradish peroxidase chromogenic substrate.

[0051] Alternatively, the antibody, attached to a surface such as amicrotiter plate or polystyrene bead, is incubated with an aliquot ofthe biological sample. MMP present in the fluid will be bound by theantibody in a manner dependent upon the concentration of MMP and theassociation constant between the two. After washing, the antibody/MMPcomplex is incubated with a second antibody specific for a differentepitope on MMP distal enough from the MMP-specific antibody binding sitesuch that stearic hindrance in binding of two antibodies simultaneouslyto MMP may be accomplished. For example, the second antibody may bespecific for a portion of the proenzyme sequence. The second antibodycan be labeled in a manner suitable for detection, such as byradioisotope, a fluorescent compound or a covalently linked enzyme. Theamount of labeled secondary antibody bound after washing away unboundsecondary antibody is proportional to the amount of MMP present in thebiological sample.

[0052] The above examples of immunoassays describe the use ofradioactively and enzymatically labeled tracers. Assays also may includeuse of fluorescent materials such as fluorescein and analogs thereof,5-dimethylaminonaphthalene-1-sulfonyl derivatives, rhodamine and analogsthereof, coumarin analogs, and phycobiliproteins such as allophycocyaninand R-phycoerythrin; phosphorescent materials such as erythrosin andeuropium; luminescent materials such as luminol and luciferin; and solssuch as gold and organic dyes. In one embodiment of the presentinvention, the biological sample is treated to remove low molecularweight contaminants.

[0053] In one embodiment of the present invention, the biological sampleis treated to remove low molecular weight contaminants, for example, bydialysis. By the term “dialysis” this invention includes any techniqueof separating the enzymes in the sample from low molecular weightcontaminants. The Examples use Spectra/Por membrane dialysis tubing witha molecular weight cut-off (MWCO) of 3,500, however other products withdifferent MWCO levels are functionally equivalent. Other productsinclude hollow fiber concentration systems consisting of regeneratedcellulose fibers (with MWCO of 6,000 or 9,000) for larger volumes; amultiple dialyzer apparatus with a sample size for one to 5 ml; andmultiple microdialyzer apparatus, convenient for samples in plates with96 wells and MWCOs at 5,000, 8,000 and 10,000, for example. Theseapparatuses are available from PGC Scientific, Gaithersburg, Md., 20898.Those with skill in the art will appreciate the utility of multipledialysis units, and especially suitable for kits for reference lab andclinic usage. Other equivalent techniques include passage through acolumn holding a resin or mixture of resins suitable to removal of lowmolecular weight materials. Resins such as BioGel (BioRad, Hercules,Calif.) and Sepharose (Pharmacia, Piscataway, N.J.) and others arewell-known to the skilled artisan. The technique of dialysis, orequivalent techniques with the same function, are intended to remove lowmolecular weight contaminants from the biological fluids. While not anessential component of the present invention, the step of removal ofsuch contaminants facilitates detection of the disorder-associatedenzymes in the biological samples.

[0054] The invention is further illustrated by the following examples,which should not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES

[0055] The following materials and methods were used throughout theseExamples, set forth below.

Materials and Methods

[0056] Urine Sample Collection and Preparation

[0057] Urine sample collection was performed as described in Moses, M.A., et al. (1998) Cancer Res. 58:1395-9, the contents of which areherein incorporated by reference in their entirety. Samples wereimmediately frozen after collection and stored frozen at −20° C. untilassay. Prior to analysis, specimens containing blood or leukocytes wereexcluded by testing for the presence of blood and leukocytes usingMultistix 9 Urinalysis Strips (Bayer, Elkhart, Ind.). The creatineconcentrations of urine samples were determined using a commercial kit(Sigma Chemical Co., St. Louis, Mo.) according to manufacturer'sinstructions.

[0058] Substrate Gel Electrophoresis

[0059] Substrate gel electrophoresis was performed based on a previouslydescribed in U.S. Ser. No. 09/469,637 with modifications. Original urinesamples (50 μl) were mixed with non-reducing sample buffer [4% sodiumdodecyl sulfate (SDS), 0.15 M Tris pH 6.8, 20% v/v glycerol, and 0.5%v/v bromphenol blue] and were separated on a 10% polyacrylamide gelcontaining 0.1% gelatin (Bio-Rad, Hercules, Calif.). Afterelectrophoresis, gels were washed twice with 2.5% Triton X-100 (15minutes/each wash). Substrate digestion was carried out by incubatingthe gel in 50 mM Tris-HCl (pH 7.6) containing 5 mM CaCl₂, 1 μM ZnCl₂, 1%Triton X-100, and 0.02% NaN₃ at 37° C. for 24hours. The gel was stainedwith 0.1% Coomassie Brilliant Blue R250 (BioRad, Hercules, Calif.), andthe location of gelatinolytic activities were detected as clear bands onthe background of a uniform blue staining.

[0060] Protein Electrophoresis and Western Blot Analysis

[0061] Urine samples were concentrated using an UltraFree-4 centrifugalfilter device with molecular weight cut off (MWCO) of 50 kDa (Millipore,Bedford, Mass.). Protein concentrations of the concentrated urinesamples were determined using the MicroBCA method (Pierce, Rockford,Ill. 61105). Equal amount of proteins (20 μg) was loaded onto 4-15%gradient gels and separated by SDS-PAGE under non-reducing conditions.Resolved proteins were electrophoretically transferred to nitrocellulosemembranes (TransBlot, Bio-Rad, Hercules, Calif.). The membranes wereblocked with 5% low fat dry milk in TBS-T (10 mM Tris, pH 7.2, 50 mMNaCl, 0.5% Tween 20) for 1 hour at room temperature, followed byincubating with primary antibody at 4° C. for 18 hours. Blots werewashed 8 times with TBS-T (5 minutes/wash) and incubated with 1:5000dilution of horseradish peroxidase (HRP) conjugated secondary antibody(Vector Laboratories, Burlingame, Calif.) diluted in TBS-T containing 3%BSA for 1 hour at room temperature. Labeled proteins were visualizedwith enhanced chemiluminescence (Amersham, Arlington Heights, Ill.).Purified polyclonal antibodies against human NGAL were used at 1:100dilution (Kjeldsen, L., et al. (1993)). Purified human neutrophilMMP-9/NGAL complex was used as positive control (CalBiochem, La Jolla,Calif.).

[0062] Immunoprecipitation

[0063] Original urine samples containing the 125 kDa MMP activity weremixed with equal volumes of RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5%sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 0.02% sodium azide).50 μl diluted urine samples were mixed with increasing amount of therabbit anti-human NGAL antibody or a control antibody. After incubatingon ice for thirty minutes, samples were mixed with 5 μl RIPA-bufferedZysorbin (ZyoMed Laboratories, South San Francisco, Calif.). Followed anadditional incubation on ice for thirty minutes, the antibody-antigencomplexes were removed with a centrifugation at 10,000 g for 5 minutes.The supernatants were subjected to substrate gel electrophoresis todetect the remaining MMP activities.

[0064] In Vitro Reconstitution of MMP-NGAL Complexes

[0065] Recombinant human proMMP-9 (Oncogene, Cambridge, Mass.) wasdiluted with gelatinase buffer [50 mM Sodium Acetate (pH=5.5) or 50 mMTris-HCl (pH=7.0, 7.6, or 8.0) containing 5 mM CaCl_(2, 1) μM ZnCl₂], toa final concentration of 10 μM. Recombinant human NGAL was purified aspreviously described and was diluted to 70 μM in the gelatinase buffer.ProMMP-9 was mixed with NGAL in a molar ratio of 1:20 and was incubatedat 37° C. for one hour. The formation of MMP-9/NGAL complex was analyzedusing substrate gel electrophoresis. ProMMP-9 and NGAL were alsoindividually diluted in normal control urine with no MMP activity. Thepossibility of MMP-9/NGAL complex formation in urine was investigated bymixing proMMP-9 and NGAL in moral ratios of 2:1, 1:5, 1:10 and 1:20.MMP-9/NGAL complex was detected using substrate gel electrophoresis.

Example 1

[0066] Substrate gel electrophoresis of MMP activities in urine samplesMMP activities contained in urine samples were assayed using substrategel electrophoresis. 50 μl of freshly thawed urine sample was used foranalysis. At least four major MMP activities were readily detected inthese urine samples, with apparent molecular mass of 200,000, 125,000,92,000, and 72,000 (FIG. 1A). The 92 kDa and the 72 kDa MMP activitieshave previously been determined to be MMP-9 and MMP-2 respectively.

[0067] The 200 kDa MMP activities is in correspondence with thepredicted molecular size of MMP-9 dimer. The identity of the 125 kDa MMPis unclear. When analyzed together with purified human MMP-9/NGALcomplex from neutrophil, the 125 kDa urinary MMP activity migrated inthe same position as that of human neutrophil MMP-9/NGAL (FIG. 2A). This125 kDa urinary MMP is an active complex of MMP-9 and NGAL. The identityof these gelatinolytic activities of being MMPs was confirmed byinhibition studies using 1,10-phenanthroline at a final concentration of10 mM (data not shown).

Example 2

[0068] Western blot analysis of urine samples with anti-NGAL antibody

[0069] To further demonstrate the identity of the 125 kDa urinary MMP asa complex of MMP-9 and NGAL, concentrated urine samples were subjectedto Western blot analysis using a purified antibody against human NGAL(Kjeldsen, L. (1993)). Under non-reducing conditions, a protein band of125 kDa was detected in urine samples containing the 125 kDa MMPactivity (FIG. 1B). Screening of urine samples from cancer patientsestablished a correlationship between the detection of MMP-9/NGALprotein complex and the presence of the 125 kDa MMP activity (FIG. 1B).Using the purified anti-NGAL antibody, a 125 kDa protein band wasconsistently detected in urine samples containing the 125 kDa MMPactivity. The antibody also detected the presence of NGAL monomer (25kDa), dimer (50 kDa), and trimer forms (75 kDa) in all of the urinesamples analyzed. The specificity of the NGAL antibody was confirmedusing purified human neutrophil MMP-9/NGAL complex. Under non-reducingconditions, the antibody recognized the 125 kDa MMP-9/NGAL complex inthe concentrated urine sample, as well as the MMP-9/NGAL complexpurified from neutrophil (FIG. 2B). In addition to the MMP-9/NGALcomplex and the NGAL monomer, dimer and trimer complexes, several minorprotein bands with approximate molecular sizes of 150 kDa were alsodetected in the concentrated urine sample. Although their identities arecurrently unclear, they are most likely to be proteins thatnon-specifically cross-reacted with anti-NGAL antibody.

Example 3

[0070] Immunoprecipitation-Zymography

[0071] To further verify the identity of the 125 kDa MMP activity inurine, anti-NGAL antibody was used to immunoprecipitate any MMPactivities that exist in the complex form with NGAL in urine. As shownin FIG. 3, anti-NGAL antibody specifically immunoprecipitated the 125kDa urinary MMP activity, in a concentration-dependent manner.Increasing amounts of the 125 kDa urinary MMP activity was removed bythe treatment with increasing amounts of anti-NGAL antibody. Whentreated with 1.0 μl of anti-NGAL antibody, the 125 kDa MMP activity wascompletely removed. The anti-NGAL antibody had no effect on any otherMMP activities, e.g., the 200 kDa MMP-9 dimer, the 92 kDa MMP-9, or the72 kDa MMP-2. The specificity of immunoprecipitation was also confirmedusing a control antibody which did not immunoprecipitate any of the MMPactivities, even at the highest concentration. The increase in MMP-2activity in the sample treated with 1.0 μl of control antibody resultedfrom endogenous MMP-2 activity contained in the serum. Taken togetherthese data support our finding that the 125 kDa MMP activity in urinesamples of cancer patients is a complex of MMP-9 and NGAL.

Example 4

[0072] Re-Constitution of MMP-9/NGAL Complex in Vitro

[0073] The formation of MMP-9/NGAL complex was first investigated usinggelatinase buffer that contains cationic ions. Recombinant humanproMMP-9 and human NGAL were first diluted in gelatinase buffers withdifferent pH values (5.5, 7.0, 7.6 and 8.0). Diluted proMMP-9 and NGALwere subsequently mixed in a molar ratio of 1:10, to finalconcentrations of 2.6 μM and 26 μM respectively. After one hourincubation at 37° C., the formation of MMP-9/NGAL complexes wasmonitored using substrate gel electrophoresis. Mixing proMMP-9 and NGALgenerated a predominant MMP activity with a molecular size ofapproximately 115 kDa (FIG. 4A). Formation of the 115 kDa MMP-9/NGALcomplex occurred in buffers with pH values ranging from 5.5 to 8.0, thepH range of normal urine. However, the size of this predominant MMPactivity is not the same as that of purified human neutrophilMMP-9/NGAL. There is a minor MMP activity of 125 kDa, observed in pH7.0, 7.6 and 8.0 buffers. The possibility of MMP-9/NGAL complexformation in urine was directly studied by diluting proMMP-9 and NGAL innormal control urine. Diluted proMMP-9 and NGAL were mixed in differentmolar ratios (proMMP-9/NGLA 2:1, 1:5, 1:10 and 1:20) and incubated at37° C. for one hour. The formation of a 115 kDa MMP-9/NGAL complex wasreadily detected in all mixing ratios (FIG. 4B). No MMP activity wasdetected in the control urine used as a diluent.

Example 5

[0074] Modulation of MMP-9 Degradation by NGAL in Vitro

[0075] The effect of NGAL on MMP-9 degradation in vitro was studied bymixing MMP-9 (0.1μ) and NGAL (1/0μ) prior to incubation. MMP-9degradation was inhibited in the presence of NGAL resulting in adecrease in the enzymatic degradation rate as evidenced by an increasein the remaining amounts of enzyme at each time point compared withMMP-9 incubated by itself. Immunodepleted NGAL had no apparentprotection of MMP-9. In the presence of increasing amount of NGAL,degradation of MMP-9 decreased and resulted in an increase in theremaining MMP-9 activity. NGAL appears to be capable of protecting MMP-9from degradation in a dose-dependent manner, resulting in thepreservation of MMP-9 activity. These data suggest a potentialregulatory role for NGAL in modulating MMP-9 activity, for example, NGALmay be involved in tumor progression via its interaction with MMP-9.

Example 6

[0076] Modulation of MMP-9 Degradation by NGAL in Cell Culture

[0077] The protective effect of NGAL on MMP-9 degradation was studied incell culture using MDA-MB-231 human breast carcinoma cells. MMP-9activity was detected in cells overexpressing NGAL (N-2 and N-5). Thus,it appeared that elevated NGAL expression resulted in an increase inMMP-9 activity. Steady state MMP-9 mRNA levels were determined usingRT-PCT analysis and no apparent differences were detected. Expressionlevels of endogenous MMP-9 inhibitor, TIMP-1, and a house-keeping gene,GAPGH, were determined and overexpression of NGAL had no apparentinfluence on mRNA levels of TIMP-1 or GAPDH. Overexpression of NGAL inhuman breast carcinoma cells resulted in an increase in MMP-9 activityindependent of changes in MMP-9 gene transcription.

Discussion

[0078] Identification of hMW enzyme complexes in the urine of cancerpatients, e.g., enzyme complexes comprising MMP-9 and NGAL, ispredictive of TRAC and is supported by the following findings: (a) the125 kDa MMP activity in urine migrates at the same position as humanneutrophil MMP-9/NGAL does; (b) anti-NGAL antibody successfully detecteda 125 kDa protein band in most of the concentrated urine samples thatcontain the 125 kDa MMP activity; (c) the same antibody was able tospecifically immunoprecipitate the 125 kDa MMP activity in urine in aconcentration-dependent manner, without affecting any other MMPactivities. Such evidence agrees with the findings described in U.S.Ser. No. 09/469,637, which is incorporated herein by reference in itsentirety, that the detection of hMW MMPs, as well as MMP-9 and MMP-2,serves as independent predictors of metastatic or organ-confinedcancers, respectively.

[0079] NGAL was first identified as a 25 kDa protein that wasco-purified with human neutrophil gelatinase (Kjeldsen, L., et al.(1993) J Biol Chem. 268: 10425-32). Binding of NGAL and MMP-9 results ina gelatinase activity of 135 kDa detected in specific granules of humanneutrophil stimulated with phorbol myristate acetate (PMA) (Kjeldsen, L.et al. (1993)). NGAL and MMP-9 are stored in specific granules, whileMMP-9 is also present independently in gelatinase granules (Morel, F.,et al., (1994) Biochim Biophys Acta. 1201: 373-80; Kjeldsen, L., et al.(1994) Blood. 83: 799-807; and Borregaard, N. and Cowland, J. B. (1997)Blood. 89: 3503-21). However, the MMP-9/NGLA complex detected in urineof cancer patients are not derived from leukocytes since we havespecifically excluded the urine samples that contain leukocytes.

[0080] Interestingly, human NGAL contains sequence similarities to mouse24p3 and rat neu/HER2/c-erbB-2 related lipocalin (NRL), bothoverexpressed in oncogene mediated cell transformation (Cowland, J. B.and Borregaard, N. (1997) Genomics. 45: 17-23; Hraba-Renevey, S., et al.(1989) Oncogene. 4: 601-8; Stoesz, S. P. and Gould, M. N. (1995)Oncogene. 11: 2233-41). Under normal conditions, expression of humanNGAL is restricted to breast, lung, trachea, and bone marrow (Cowland,J. B. and Borregaard, N. (1997) Genomics. 45: 17-23; Stoesz, S. P., etal. (1998) Int. J Cancer. 79: 565-72). However, elevated levels of NGALexpression has been observed in human breast tumors as well as inadenocarcinomas of lung, colon and pancreas (Stoesz (1998); Friedl, A.,et al.(1999) Histochem J. 31: 433-41). An increased production of NGALcan be closely associated with cancer disease status, which subsequentlycontribute to the elevated levels of MMP-9/NGAL complex in urine. Thiscomplex can be detected with substrate gel electrophoresis as well asantibody-based assays. As described in U.S. Ser. No. 09/469,637, thepresence of the 125 kDa MMP activity in urine can serve as anindependent multivariate predictor of cancer metastasis, theidentification of this activity as MMP-9/NGAL complex will facilitatethe development of a non-invasive prognosis tool to assess diseasestatus of various cancers.

[0081] The origin of the 125 kDa MMP-9/NGAL activity in urine of cancerpatients remains unclear. Given that the glomerular filtration limit isonly 45 kDa, it is unlikely that this large protein complex is directlyfiltered from serum into urine. The possibility that MMP-9/NGAL complexforms after each component was separately filtrated into urine wasinvestigated using in vitro reconstitution assay. The resultsdemonstrate the feasibility of MMP-9/NGAL complex formation ingelatinase buffers with different pH values, as well as, in normalurine. Therefore, it is likely that MMP-9 and NGAL are separatelyexecuted into urine where they form the 125 kDa MMP-9/NGAL complex.

[0082] The existence of MMP-9 and NGAL complex in urine was supported bya recent independent study (Monier, F., Clin Chim Acta. 299: 11-23,2000). Under reducing conditions, MMP-9 and NGAL were separatelydetected in a continuous-elution electrophoresis fraction that containsa 115 kDa gelatinase activity. The detection of MMP-9 and NGAL in thesame fraction shows the observed 115 kDa gelatinase activity as acomplex of MMP-9 and NGAL.

[0083] Recent studies have also confirmed that NGAL appears to exert aprotective effect on MMP-9 and prevents MMP-9 from degradation both invitro and in cells. Examples 5 and 6 suggest that the MMP-9-NGAL complexlikely plays an active role in tumor progression.

Equivalents

[0084] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. A non-invasive method for facilitating thediagnosis of a subject for a tissue remodelling-associated condition,comprising: obtaining a biological sample from a subject; detecting ahigh molecular weight enzyme complex in the biological sample; andcorrelating the presence or absence of the high molecular weight enzymecomplex with the presence or absence of a tissue remodelling-associatedcondition, thereby facilitating the diagnosis of the subject for atissue remodelling-associated condition.
 2. The method of claim 1,wherein the tissue remodelling-associated condition is cancer.
 3. Themethod of claim 1, wherein the tissue remodelling-associated conditionis an arthritic condition, an obstructive condition, or a degenerativecondition.
 4. The method of claim 2, wherein the cancer isorgan-confined prostate cancer.
 5. The method of claim 2, wherein thecancer is metastatic prostate cancer.
 6. The method of claim 2, whereinthe cancer is in cells of epithelial origin.
 7. The method of claim 6,wherein the cancer is selected from the group consisting of cancers ofthe nervous system, breast, retina, lung, skin, kidney, liver, pancreas,genito-urinary tract, and gastrointestinal tract.
 8. The method of claim2, wherein the cancer appears in cells of mesodermal origin.
 9. Themethod of claim 2, wherein the cancer appears in cells of endodermalorigin.
 10. The method of claim 2, wherein the cancer affects cells ofbone or of hematopoietic origin.
 11. The method of claim 1, wherein thehigh molecular weight enzyme complex comprises a protease.
 12. Themethod of claim 11, wherein the protease is a serine protease.
 13. Themethod of claim 11, wherein the protease is a matrix metalloproteinase.14. The method of claim 13, wherein the matrix metalloproteinase is anMMP-9.
 15. The method of claim 1, wherein the high molecular weightenzyme complex comprises a lipocalin.
 16. The method of claim 15,wherein the lipocalin is NGAL.
 17. The method of claim 15, wherein theenzyme complex comprises a TIMP.
 18. The method of claim 17, wherein theTIMP is TIMP-1.
 19. The method of claim 1, wherein the high molecularweight enzyme complex comprises an enzyme complexed with itself to forma multimer.
 20. The method of claim 19, wherein the multimer is a dimeror a trimer.
 21. The method of claim 19, wherein the multimer is furthercomplexed with a lipocalin.
 22. The method of claim 21, wherein thelipocalin is NGAL.
 23. The method of claim 21, wherein the multimer isfurther complexed with a TIMP.
 24. The method of claim 23, wherein theTIMP is TIMP-1.
 25. The method of claim 1, wherein the molecular weightof the enzyme complex is approximately 150 kDa.
 26. The method of claim1, wherein the molecular weight of the enzyme complex is approximately115 to approximately 125 kDa.
 27. A non-invasive method for facilitatingthe diagnosis of a subject for a tissue remodelling-associatedcondition, comprising: obtaining a biological sample from a subject;detecting lipocalin in the biological sample; and correlating thepresence or absence of the lipocalin with the presence or absence of atissue remodelling-associated condition, thereby facilitating thediagnosis of the subject for a tissue remodelling-associated condition.28. The method of claim 27, wherein the tissue remodelling-associatedcondition is cancer.
 29. The method of claim 27, wherein the tissueremodelling-associated condition is an arthritic condition, anobstructive condition, or a degenerative condition.
 30. The method ofclaim 28, wherein the cancer is organ-confined prostate cancer.
 31. Themethod of claim 28, wherein the cancer is metastatic prostate cancer.32. The method of claim 28, wherein the cancer is in cells of epithelialorigin.
 33. The method of claim 32, wherein the cancer is selected fromthe group consisting of cancers of the nervous system, breast, retina,lung, skin, kidney, liver, pancreas, genito-urinary tract, andgastrointestinal tract.
 34. The method of claim 28, wherein the cancerappears in cells of mesodermal origin.
 35. The method of claim 28,wherein the cancer appears in cells of endodermal origin.
 36. The methodof claim 28, wherein the cancer affects cells of bone or ofhematopoietic origin.
 37. The method of claim 27, wherein the lipocalinis NGAL.
 38. The method of claim 27, wherein the lipocalin exists as amultimer.
 39. The method of claim 38, wherein the multimer is a dimer ora trimer.
 40. The method of any one of claims 1 or 27, wherein thebiological sample is urine.
 41. The method of claim 40, furthercomprising removal of low molecular weight contaminants from the urineprior to the detection step.
 42. The method of claim 41, wherein theurine is dialyzed.
 43. The method of claims 1 or 27, wherein the enzymeis detected by an electrophoretic pattern.
 44. The method of claim 43,wherein the electrophoretic pattern is a zymogram comprising asubstrate.
 45. The method of claim 44, wherein the zymogram substrate isselected from the group consisting of gelatin, casein, fibronectin,vitronectin, plasmin, plasminogen, type IV collagen, and a derivative oftype IV collagen.
 46. The method of any one of claims 1 or 27, whereinthe enzyme is detected immunochemically.
 47. The method of claim 46,wherein the enzyme is detected by a radio-immunoassay.
 48. The method ofclaim 46, wherein the enzyme is detected by an enzyme-linkedimmunosorbant assay.
 49. A kit for facilitating the diagnosis andprognosis of a tissue remodelling-associated condition, comprising: acontainer having a reagent for detecting a high molecular weight enzymecomplex in a biological sample; and instructions for using said reagentfor detecting the high molecular weight enzyme complex for facilitatingthe diagnosis and prognosis of a tissue remodelling-associatedcondition.
 50. The kit of claim 49, wherein the tissueremodelling-associated condition is cancer.
 51. The kit of claim 49,wherein the tissue remodelling-associated condition is an arthriticcondition, an obstructive condition, or a degenerative condition. 52.The kit of claim 50, wherein the cancer is organ-confined prostatecancer.
 53. The kit of claim 50, wherein the cancer is metastaticprostate cancer.
 54. The kit of claim 50, wherein the cancer is in cellsof epithelial origin.
 55. The kit of claim 54, wherein the cancer isselected from the group consisting of cancers of the nervous system,breast, retina, lung, skin, kidney, liver, pancreas, genito-urinarytract, and gastrointestinal tract.
 56. The kit of claim 50, wherein thecancer appears in cells of mesodermal origin.
 57. The kit of claim 50,wherein the cancer appears in cells of endodermal origin.
 58. The kit ofclaim 50, wherein the cancer affects cells of b one or of hematopoieticorigin.
 59. The kit of claim 49, wherein the high molecular weightenzyme complex comprises a protease.
 60. The kit of claim 59, whereinthe protease is a serine protease.
 61. The kit of claim 59, wherein theprotease is a matrix metalloproteinase.
 62. The kit of claim 61, whereinthe matrix metalloproteinase is an MMP-9.
 63. The kit of claim 49,wherein the high molecular weight enzyme complex comprises a lipocalin.64. The kit of claim 63, wherein the lipocalin is NGAL.
 65. The kit ofclaim 63, wherein the enzyme complex comprises a TIMP.
 66. The kit ofclaim 65, wherein the TIMP is TIMP-1.
 67. The kit of claim 49, whereinthe high molecular weight enzyme complex comprises an enzyme complexedwith itself to form a multimer.
 68. The kit of claim 67, wherein themultimer is a dimer or a trimer.
 69. The kit of claim 67, wherein themultimer is further complexed with a lipocalin.
 70. The kit of claim 69,wherein the lipocalin is NGAL.
 71. The kit of claim 69, wherein themultimer is further complexed with a TIMP.
 72. The kit of claim 71,wherein the TIMP is TIMP-1.
 73. The kit of claim 49, wherein themolecular weight of the enzyme complex is approximately 150 kDa.
 74. Thekit of claim 49, wherein the molecular weight of the enzyme complex isapproximately 115 to approximately 125 kDa.
 75. The kit of claim 49,wherein the biological sample is urine.
 76. The kit of claim 75, furthercomprising an apparatus for separating urine into components for removalof low molecular weight contaminants.
 77. The method or kit of any oneof claims 1, 27, or 49, wherein the high molecular weight enzyme complexdoes not have a molecular weight of 115 kDa.
 78. The method or kit ofany one of claims 1, 27, or 49, wherein the high molecular weight enzymecomplex does not comprise a progelatinase B enzyme.
 79. The method orkit of any one of claims 1, 27, or 49, wherein the high molecular weightenzyme complex does not comprise NGAL.
 80. The method or kit of any oneof claims 1, 27, or 49, wherein the high molecular weight enzyme complexdoes not comprise a progelatinase B enzyme associated with NGAL.